Individual neurons from different parts of the brain may be taken from animals and cultivated in biologically compatible environments. However, if an ex vivo neural network could be established, it could then be studied by stimulating neurons with electric signals and observing how the live network reacts and modifies itself. This could bring us closer to understanding how a neural network modifies its structure during the learning phase and the rules that govern the way synapses and neurites grow. The analysis of the electro-physiological activity of the neurons in the neural network may allow us to develop artificial prostheses for by-passing injured zones and restore brain functionality, or to realize neuro-diagnostic tools for monitoring the reaction of biological neurons to selected chemical species or newly developed drugs. But in order to reach this objective, we need suitable devices for maintaining a live neural network with electrical stimulation and detection capabilities.
Specifically, we need a device that can spatially arrange a plurality of live neurons at individual fixed positions with reliable and durable electrical coupling to stimulation and detection circuitry. The device should allow the confined neurons to grow and develop synaptic connections for creating a neural network and communication. For applying and detecting electric signals there must be means for ensuring a stable contact of the body of each spatially confined neuron to an electrode or with a functionally equivalent electrical coupling element, connected to a circuit for stimulating neurons and for detecting electrical signals exchanged among them.
There are many research teams that study neural activity by stimulating and recording electrical signals coming from distinct zones of a nervous tissue (hippocampus, cortex etc.), but the main difficulty is electrically coupling external stimulation and sensing circuitries to the neurons.
This is currently established through coupling elements of two kinds: invasive interfaces (electrodes are implanted “in vivo” in a nervous tissue); and noninvasive interfaces (where neural tissue contacts a silicon chip substrate establishing an electrical coupling with an embedded electrode).
The main drawback of invasive interfaces, typically employing intra-cellular electrodes, is the risk of irremediably damaging the cell during experiments. Moreover, it is very difficult to use more than two electrodes at the same time for stimulating the neural network because the actuators used for correctly positioning the microelectrodes are very cumbersome.
In order to overcome this problem, effective noninvasive interfaces for coupling neurons to external electronic devices are being earnestly searched and developed. For example, Dr. Roberta Diaz Brinton grew rat hippocampus neurons on a silicon substrate at the University of Southern California. The objective of his experiment was to use hybrid brain-silicon systems for studying the processes by which a brain carries out complex operations, such as pattern recognition.
According to this methodology, dissociated neurons were placed on a silicon test substrate having an array of electrodes and coated with a material to which the neural cells could stably adhere. The neurons fixed themselves to the silicon substrate and grew, sprouting processes and synaptic connections with other neurons. The growth of the neurons colonies was directable: by using masks, it was possible to predefine paths along which growing neurites would extend. The electrodes onto which the neurons were cultivated were used both for stimulating neurons as well as for monitoring their electrical activity.
At Caltech (California Institute of Technology), a device called a “neurochip” has been realized in which a network of live brain cells was connected through electrodes on a silicon chip to stimulation and detection circuitry [1] [2]. Neurons having a maximum diameter of about 15 μm were taken from the superior cervical ganglion (SCG) of a rat. The “neurochip” had three main features: a well formed in the silicon substrate into which a neural cell was confined, an overhanging grillwork for trapping the cell body inside the well, and an electrode in contact with the trapped neuron.
The disclosed “neurochip” was composed of sixteen trapezoidal wells, closed at the top by an overhanging grillwork of patterned heavily doped silicon, constituting a 4×4 array, realized on a silicon wafer by photolithography and “micromachining” of the silicon crystal. On the bottom face of the silicon wafer, a predefined 4×4 array of gold electrodes that closed the bottom of the wells provided a stable electrical contact with the entrapped cell bodies. The surface of the electrodes at the bottom of the wells was covered with platinum black for reducing contact impedance with the body of the neural cell.
The entrapment grillwork was designed to permit the introduction of an embryonic neural cell into each well and prevent that cell from escaping. At the same time, the grillwork allowed neurites to sprout through the apertures of the grillwork and connect to other neurons to form a live neural network.
A variety of different grillwork patterns have been tested to prevent cell escape. In a recent release of the neurochip, depicted in FIG. 1, a MEMS structure forms a sort of canopy above the well. The overhanging grillwork above the etched cavity in the silicon substrate has openings through which neurites may sprout.
FIG. 2 is a SEM (Scanning Electron Microscope) picture of the grillwork and a cross sectional schematic of the trapping well closed by the retention grillwork showing the openings through which the neuron grows and eventually develops its neurites.
The height of the openings through the grillwork (micro tunnels) depends on the thickness of the patterned nitride layer that constitutes the overhanging grillwork. An appropriate choice of the dimensions of these micro tunnels allows neurons to grow out of the well cavity, but preventing their escape. Experiments have shown that the crucial parameter in preventing neuron escape through the growth “microtunnels” is not their breadth but their extension (length), that is the thickness of the grillwork nitride layer.
However, reliable entrapment of neurons by means of an insurmountable overhanging grillwork that obstructs the well opening have the disadvantage of not allowing the replacement of dead cells without irreparably damaging the confining device.
At the “Max Planck Institute for Biochemistry” in Munich, Germany, Peter Fromherz and Gunther Zech carried out experiments on neurons of “Lymnaea Stagnalis” [3] that, being an invertebrate (a kind of slug), has neurons with a relatively large body that contact the underlying interfacing substrate very well. These neurons, even in small numbers, were capable of reproducing normal biological functions.
The neurons were cultivated onto a silicon chip, shown in FIG. 3. The letters S, G and D indicate the source, gate and drain terminals, respectively, of an integrated Filed Effect Transistor (FET). The white scale line is 20 μm long.
The chip was covered with a layer of silicon oxide for preventing electrochemical phenomena at the neuron-substrate contact surface and also for creating a homogeneous and inert rest surface for the neurons. Neurons were confined on the silicon substrate by means of a polyamide picket fence.
In order to ensure a noninvasive neuron-silicon interface, a two-way electrical coupling was established by means of the FET and a stimulator (ST). The stimulator (ST) was substantially constituted by a P doped zone onto an N doped silicon region covered by a thin layer of silicon oxide. The source and drain terminals of the transistor were realized in distinct P doped regions with a gate area covered by a thin gate oxide layer not topped by any metal gate electrode layer.
The stimulator (ST) provided a capacitive coupling between the chip and the neuron, while the transistor, integrated in the silicon under the neuron body, sensed the extra-cellular voltage.
FIG. 4 shows a neuron grown on the device of FIG. 3 after having been cultivated for three days. FIG. 5 is a microscope image of neural cells (dark circles) each confined by a six picket fences of polyamide. Some pickets of adjacent fences have combined, forming a single picket of elongated cross-section. The light gray lines that originate from the cells are neurites grown on the surfaces of the chip that connect the neurons among them. The radially extending straight lines are the traces of the connecting metal lines of the stimulators and sensing transistors.
By electrically exciting a neuron (via the stimulator), an electrical activity is induced in another neuron of the network that modulates the current in the transistor underneath it, thus amplifying the tenuous electrical signal. Such a detected variation of potential indicates that an electrical synapse has been established between the two neurons.
Notwithstanding the effectiveness of these devices, reliable and durable spatial confinement of the neural cells is precarious because the neurons tend to escape the picket fence.
The recurrent problem in designing these devices is to reliably prevent cell escape, but still allow cell growth, and to reliable couple the cells to electrodes (or alternate stimulation and detection means), preferably on an easily micromachinable material such as a monocrystalline silicon chip (wafer). The fact that the confined cell may not properly adhere to the surface of the substrate and thus fail to remain in stable contact with the electrical stimulation and detection elements may impede the stimulation and the monitoring of exchanged electrical messages. The known interfacing structures discussed above represent the best compromise currently available, but better devices are still needed.